Improving the predictive power of xenograft and syngeneic anti-tumour studies using mice humanised for pathways of drug metabolism

Drug development is an expensive and time-consuming process, with only a small fraction of drugs gaining regulatory approval from the often many thousands of candidates identified during target validation. Once a lead compound has been identified and optimised, they are subject to intensive pre-clinical research to determine their pharmacodynamic, pharmacokinetic and toxicological properties, procedures which inevitably involve significant numbers of animals - mainly mice and rats, but also dogs and monkeys in much smaller numbers and for specific types of drug candidates. Many compounds that emerge from this process, having been shown to be safe and efficacious in pre-clinical studies, subsequently fail to replicate this outcome in clinical trials, therefore wasting time, money and, most importantly, animals. Due to high rates of metabolism and a differing spectrum of metabolites (some pharmacologically active) in rodents, species differences in drug metabolism can be a major impediment to drug discovery programmes and confound the extrapolation of animal data to humans. To circumvent this, we have developed a complex transgenic mouse model – 8HUM - which faithfully replicates human Phase I drug metabolism (and its regulation), and which will generate more human-relevant data from fewer animals in a pre-clinical setting and reduce attrition in the clinic. One key area for the pre-clinical application of animals in an oncology setting – almost exclusively mice - is their use in anti-tumour studies. We now further demonstrate the utility of the 8HUM mouse using a murine melanoma cell line as a syngeneic tumour and also present an immunodeficient version 8HUM_Rag2 -/- - for use in xenograft studies. These models will be of significant benefit not only to Pharma for pre-clinical drug development work, but also throughout the drug efficacy, toxicology, pharmacology, and drug metabolism communities, where fewer animals will be needed to generate more human-relevant data

Improving the predictive power of xenograft and syngeneic anti-tumour studies using mice humanised for pathways of drug metabolism [version 2; peer review: 2 approved]

Drug development is an expensive and time-consuming process, with only a small fraction of drugs gaining regulatory approval from the often many thousands of candidates identified during target validation. Once a lead compound has been identified and optimised, they are subject to intensive pre-clinical research to determine their pharmacodynamic, pharmacokinetic and toxicological properties, procedures which inevitably involve significant numbers of animals - mainly mice and rats, but also dogs and monkeys in much smaller numbers and for specific types of drug candidates. Many compounds that emerge from this process, having been shown to be safe and efficacious in pre-clinical studies, subsequently fail to replicate this outcome in clinical trials, therefore wasting time, money and, most importantly, animals. Due to high rates of metabolism and a differing spectrum of metabolites (some pharmacologically active) in rodents, species differences in drug metabolism can be a major impediment to drug discovery programmes and confound the extrapolation of animal data to humans. To circumvent this, we have developed a complex transgenic mouse model – 8HUM - which faithfully replicates human Phase I drug metabolism (and its regulation), and which will generate more human-relevant data from fewer animals in a pre-clinical setting and reduce attrition in the clinic. One key area for the pre-clinical application of animals in an oncology setting – almost exclusively mice - is their use in anti-tumour studies. We now further demonstrate the utility of the 8HUM mouse using a murine melanoma cell line as a syngeneic tumour and also present an immunodeficient version 8HUM_Rag2 -/- - for use in xenograft studies. These models will be of significant benefit not only to Pharma for pre-clinical drug development work, but also throughout the drug efficacy, toxicology, pharmacology, and drug metabolism communities, where fewer animals will be needed to generate more human-relevant data

How Much Rugby is Too Much? A Seven-Season Prospective Cohort Study of Match Exposure and Injury Risk in Professional Rugby Union Players.

INTRODUCTION: Numerous studies have documented the incidence and nature of injuries in professional rugby union, but few have identified specific risk factors for injury in this population using appropriate statistical methods. In particular, little is known about the role of previous short-term or longer-term match exposures in current injury risk in this setting. OBJECTIVES: Our objective was to investigate the influence that match exposure has upon injury risk in rugby union. METHOD: We conducted a seven-season (2006/7-2012/13) prospective cohort study of time-loss injuries in 1253 English premiership professional players. Players' 12-month match exposure (number of matches a player was involved in for ≥20 min in the preceding 12 months) and 1-month match exposure (number of full-game equivalent [FGE] matches in preceding 30 days) were assessed as risk factors for injury using a nested frailty model and magnitude-based inferences. RESULTS: The 12-month match exposure was associated with injury risk in a non-linear fashion; players who had been involved in fewer than ≈15 or more than ≈35 matches over the preceding 12-month period were more susceptible to injury. Monthly match exposure was linearly associated with injury risk (hazard ratio [HR]: 1.14 per 2 standard deviation [3.2 FGE] increase, 90% confidence interval [CI] 1.08-1.20; likely harmful), although this effect was substantially attenuated for players in the upper quartile for 12-month match exposures (>28 matches). CONCLUSION: A player's accumulated (12-month) and recent (1-month) match exposure substantially influences their current injury risk. Careful attention should be paid to planning the workloads and monitoring the responses of players involved in: (1) a high (>≈35) number of matches in the previous year, (2) a low (<≈15) number of matches in the previous year, and (3) a low-moderate number of matches in previous year but who have played intensively in the recent past. These findings make a major contribution to evidence-based policy decisions regarding match workload limits in professional rugby union

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Conditional deletion of cytochrome P450 oxidoreductase in the liver and gastrointestinal tract: A new model for studying the functions of the P450 system

We have previously described a mouse model, where hepatic cytochrome P450 oxidoreductase (POR) expression has been deleted, resulting in almost complete ablation of hepatic P450 function [Hepatic P450 Reductase Null (HRN)]. HRN mice grow normally but develop fatty livers, and they have increased cytochrome P450 levels. Associated with the hepatic lipid accumulation are significant changes in the expression of genes controlling lipid homeostasis. We have characterized this model extensively and demonstrated its value in drug efficiency testing, in toxicokinetics, and in evaluating the role of the hepatic P450 system in drug pharmacokinetics. To extend the deletion of POR, and P450 inactivation, to other tissues, and to develop the utility of this model, we have generated a mouse where POR can be deleted conditionally in the liver and gastrointestinal tract using the rat cytochrome P450 CYP1A1 promoter to drive Cre recombinase expression. Administration of the CYP1A1 inducers tetrachlorodibenzo-p-dioxin or β-naphthoflavone resulted in both hepatic and gastrointestinal deletion of POR, whereas administration of 3-methylcholanthrene resulted specifically in loss of hepatic POR expression. In all cases, the resulting hepatic phenotype seemed identical to that of the HRN model, including increased cytochrome P450 expression. Hepatic deletion of POR and the subsequent increase in P450 expression were dependent on inducer dose, with maximal POR deletion occurring at a single dose of 3-methylcholanthrene of 40 mg/kg. This model provides a powerful approach for studying the functions of POR as well as in the evaluation of the role of hepatic and gastrointestinal P450s in drug deposition and chemical toxicity

<i>In vivo</i> regulation of human glutathione transferase GSTP by chemopreventive agents

Relatively little progress has been made in determining the in vivo regulation of glutathione S-transferase P (GSTP), particularly the human enzyme hGSTP1, despite being identified as a significant factor in carcinogenesis and development of drug resistance in tumor cell lines. Here we report the characterization of a transgenic reporter mouse that reveals how hGSTP1 is regulated in vivo by chemopreventive agents. Basal expression was found in crypts and villi of the small and large intestine, bronchiolar epithelial cells, the epidermis and hair follicles, gall bladder epithelium, choroid plexus and biliary epithelium. Expression was induced in different tissues by the antioxidant chemopreventive agents ethoxyquin (EQ) and butylated hydroxyanisole (BHA). However, genetic deletion of the Nrf2 transcription factor, which directs central genetic programs of detoxification and protection against oxidative stress, increased rather than attenuated GSTP1 expression. In vitro investigations with mouse embryonic fibroblasts revealed factor(s) in addition to Nrf2 that control the expression of GSTP1, offering further insights into regulation. The new reporter mouse described here provides a useful tool to gain deeper insights into the mechanisms of action of chemopreventive compounds and other environmental agents